Radiopharmaceutical formulations

ABSTRACT

Radiopharmaceutical compounds are disclosed having a radionuclide chelating moiety and a targeting ligand, and optionally a linker. Formulations of compositions useful for making the radiopharmaceutical compounds contain a chelating ligand, a reducing agent, an exchange ligand and a stabilizer.

This application is the national stage application of correspondinginternational application number PCT/US03/13936 filed May 5, 2003 whichclaims priority to and the benefit of U.S. Provisional Application No.60/377,454, filed May 3, 2002, all of which are hereby incorporated byreference.

FIELD OF THE INVENTION

The invention relates to formulations for radiopharmaceuticalscomprising radionuclide chelators.

BACKGROUND OF THE INVENTION

Targeted radiopharmaceuticals resolve an image of diagnostic interest ordeliver a therapeutic radioisotope to an area of interest by binding orlocalizing selectively to a site within the body. For various diagnosticand therapeutic applications, chelators that bind a metal radionuclideand are linked to a targeting molecule have been employed with varyingdegrees of success. Such chelators often have a region incorporatingfour or more donor atoms that form five- or six-membered ringsappropriate for high affinity radionuclide binding. Typical metalradionuclides used for diagnostic imaging agents include ^(99m)Tc, ⁶⁴Cu,⁶⁷Cu, ⁹⁷Ru, ¹⁰⁹Pd, ¹⁹⁸Au, ¹⁹⁹Au, ¹¹¹In, in their various chlorides,oxides or nitrides. Typical metal radionuclides used forradiotherapeutic applications include ¹⁸⁶Re, ¹⁸⁸Re, ¹¹¹In, ¹⁶⁶Ho, ¹⁰⁵Rh,¹⁴⁹Pm, ¹⁵³Sm, ¹⁷⁷Lu, ⁹⁰y, ²⁰³Pb, ²¹²Pb and ²¹²Bi in their variouschlorides, oxides or nitrides. Numerous types of molecules have beenemployed as targeting molecules, including polyclonal and monoclonalantibodies and fragments thereof, proteins and peptides, especiallythose capable of binding with specificity to cell surface receptors(generally referred to as “receptor-binding ligands”). When labelingpeptide and protein-based targeting agents, the chelator is ideally alsopeptide-based, so that the chelator-targeting molecule conjugate can besynthesized using peptide synthesis techniques. For example, U.S. Pat.Nos. 5,662,885; 5,780,006; and 5,976,495 (each of which is incorporatedherein by reference in its entirety) disclose chelators that bind metalradionuclides, can be coupled to targeting agents capable of localizingat body sites of diagnostic and therapeutic interest, and are peptideanalogs designed structurally to present an N₃S configuration capable ofbinding oxo, dioxo and nitrido ions of, for example, ^(99m)Tc and^(186/188)Re. Moreover, peptidic cores that chelate isotopes of Tc andother diagnostic and therapeutic radionuclides are known; most of thiswork has focused on using peptides derived from natural amino acids ofthe form NH₂CHRCOOH.

Despite the many advances in diagnostic imaging, several obstacles areroutinely encountered in this field. One of the key problems is thedevelopment of useful formulations for the preparation of the targetedradiopharmaceutical. One problem frequently encountered is that manyformulations require a high concentration of the targeted chelatingligand in reactions for the preparation of a targetedradiopharmaceutical to assure high yields of the desired complex. Theseformulations produce radiopharmaceutical preparations with significantamounts of “free” targeted chelating ligand that has not been chelatedto the radiometal. For many applications this “free” ligand isundesirable; thus, it must be separated from the labeled ligand usingchromatographic techniques such as High Pressure Liquid Chromatography(HPLC) prior to further use. This is often necessary where the targetingmolecule attached to the chelator is, for example, an agonist, andexhibits biological activity when it binds to a target receptor. Suchbiological activity is undesirable in a diagnostic compound and may alsobe undesirable in a therapeutic one. In addition, receptor-bindingligands that are not complexed with a radionuclide may compete at thetarget receptor with those ligands that are complexed with aradionuclide, resulting in poor targeting of the radionuclide complexand poor diagnostic or therapeutic characteristics.

The targeted radiopharmaceutical with the structure shown in FIG. 1 isan example. The uncomplexed receptor binding ligand (or targetedchelating ligand) used to make this complex contains a tripeptide N₃Schelator [(N-(Me₂)-Gly-Ser-Cys(Acm)] that forms a complex with Tc(V)O,losing the acetamidomethyl (Acm) protecting group in the process. Thechelator sequence is linked to the N-terminus of an octapeptidetargeting molecule, pGlu-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ derived frombombesin, via a linker of Gly-aminovaleric acid. Both this ligand andthe Tc complex formed from it are agonists that are known to bind toGastrin Releasing Peptide (GRP) Receptors with high affinity. See, e.g.,U.S. Pat. No. 6,200,546, which is hereby incorporated by reference inits entirety. Clinical studies that were performed with this compound,e.g. those described by Van de Wiele et al. (European Journal of NuclearMedicine, Vol. 27, No. 11, 2000 p. 1694 (which is hereby incorporated byreference in its entirety) were prepared using the following prototype4-vial kit. To each of 2 vials, each containing 100 μg of ligand wasadded 0.1 mL of stannous chloride (2 mM), 0.1 mL of sodium gluconate (60mM), 1850-2035 MBq (50-55 mCi) of ^(99m)TcO₄— in 0.3 mL of 0.9% sodiumchloride, and 0.5 mL of sodium chloride. After 35 min. in a boilingwater bath, the pooled reaction mixtures were injected on an HPLC systemand purified in order to separate labeled from unlabelled peptide,followed by terminal sterilization. The overall yield from thisradiosynthesis was ˜30%, with a radiochemical purity after purificationof >90%. The purified compound could be stored at 4° C. for only up to 2hours. This procedure, although useful to provide initial evidence ofthe potential clinical utility of the compound, is not commerciallyviable because it uses a 4-vial frozen kit, requires preparative HPLC(i.e., instrumentation that is not typically available in nuclearmedicine units) to remove excess ligand prior to injection, and does notprovide a sterile product except by terminal sterilization. In addition,it uses >100 mCi of ^(99m)TcO₄ ⁻ to make a patient dose, requires theuse of generator eluant that is 2 hours old or less, and the purifiedproduct can only be used for 2 hours after isolation.

It would be highly advantageous to have a formulation for thepreparation of this and other receptor-binding targetedradiopharmaceuticals that, among other things, can be used clinically(e.g., directly injected) without the need for HPLC purification. Thepresent invention addresses this need and other problems in the art.

SUMMARY OF THE INVENTION

The present invention features radiopharmaceutical formulations usefulin diagnostic imaging and/or radiotherapy. In one aspect, the inventionfeatures radiopharmaceutical formulations containing targeted chelatingligands having the structure of Formula I:

wherein R¹ is H or is a linear or branched, saturated or unsaturatedC1-4 alkyl chain that is optionally interrupted by one or twoheteroatoms selected from N, O and S; and is optionally substituted byat least one group selected from halogen, hydroxyl, amino, carboxyl,C1-4 alkyl, aryl and C(O)Z;

R² is a substituent defined by R¹; or

R¹ and R² may together form a 5- to 8-membered saturated or unsaturatedheterocyclic ring optionally substituted by at least on group selectedfrom halogen, hydroxyl, amino, carboxyl, C1-4 alkyl, aryl and C(O)Z;

R³ is —H, —CH₂OH, or -t-butyl;

L is optional and is a linking group; and

Z is a targeting molecule.

In preferred embodiments, the formulation comprises compositions inwhich both R¹ and R² are methyl. In a more preferred embodiment both R¹and R² are methyl and Z is a bombesin analog, most preferably a bombesinanalog that is an agonist, such as those disclosed in U.S. Pat. No.6,200,546, incorporated by reference herein in its entirety.

Unlike many prior compositions for the preparation of targetedradiopharmaceuticals, the radiopharmaceutical formulations of theinvention utilize a small amount of chelator/targeting moleculeconjugate (e.g., compounds of Formula I). Indeed, in a preferredembodiment, radiopharmaceutical formulations of the invention areprovided that contain between about 2 to about 10 μg of a targetedchelating ligand of Formula 1, thus reducing the risk of unwantedbiological activity or physiological effects resulting from theadministration of, e.g., a biologically active peptide.

The radiopharmaceutical formulations of the invention further includethe components described in detail herein (e.g. reducing agent, exchangeligand, stabilizer, solubilizer, diluent, etc.) In a preferredembodiment, the formulation of the invention includes Compound 1,SnCl₂.2H₂O, gluconic acid (sodium salt), gentisic acid (sodium salt),ethanol and optionally hydroxypropyl-γ-cyclodextrin, or pharmaceuticallyacceptable salts thereof.

The invention also encompasses kits for the preparation of targetedradiopharmaceuticals that contain the radiopharmaceutical formulationsof the invention. Preferred kits include the uncomplexed targetedchelating ligand, the reducing agent, the exchange ligand, thestabilizer(s) and any solubilizers in one or more vials, and the diluentin a separate vial. A particularly preferred kit of the inventionincludes two vials. The first vial comprises: Compound 1, SnCl₂.2H₂O,gluconic acid (sodium salt), gentisic acid (sodium salt), and optionallyhydroxypropyl-γ-cyclodextrin. The second vial comprises ethanol, andoptionally water and/or normal saline.

In a preferred embodiment, radiopharmaceutical formulations of theinvention have a radiochemical purity value (RCP) that is greater thanor equal to 90% within 15 min. after reconstitution. In anotherpreferred embodiment, radiopharmaceutical formulations of the inventionhave a radiochemical purity value that is greater than or equal to 90%at six hours after reconstitution. Radiopharmaceuticals prepared fromformulations of the invention may be injected directly into a subjectwithout any purification. In addition, such radiopharmaceuticals may beprepared at room temperature.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the chemical structure of the ^(99m)Tc complex of Compound1, a preferred targeted chelating ligand used in formulations of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

Radiopharmaceutical formulations of the present invention havesignificant advantages over previous formulations. These formulationscontain significantly less targeted chelator than prior artformulations. Indeed the formulations of the invention contain less than10 μg of targeted chelator per mL of diluent. Preferably, aradiopharmaceutical formulation of the invention contains less thanabout 5 μg of targeted chelator per mL of diluent. Most preferably, aradiopharmaceutical formulation of the invention contains about 4 μg oftargeted chelator per mL of diluent, with a total reconstitution volumebetween about 0.5 mL and about 1 mL, most preferably 1.0 mL. Inparticular, the present invention provides radiopharmaceuticalformulations that may be prepared and administered to a subject withoutany purification. In addition, the radiopharmaceutical formulations ofthe invention have a radiochemical purity (RCP) of greater than or equalto 90% at about 15 minutes after preparation (i.e., reconstitution).Most preferred radiopharmaceutical formulations of the invention have aRCP that is greater than or equal to about 90% at about six hours afterpreparation.

The diluent used to reconstitute the radiopharmaceutical formulations ofthe present invention may be any combination of water, normal salineand/or ethanol. Preferably, the diluent is a mixture of water, normalsaline and ethanol in which the percentage of ethanol ranges from about20% to about 40% (v/v), most preferably about 30%. The pH of theformulation after reconstitution is between 2.8 and 4.0, most preferablyabout pH 3.0.

Radiopharmaceuticals of the present invention may be prepared by areaction with a reducing agent able to reduce the radionuclide (which isin an oxidized state) to a reduced state that can coordinate with theligand, such as a stannous source, sodium borohydride, Cu(I) salts,formamidine sulphinic acid and the like. The preferred reductant is astannous salt such as stannous chloride, stannous fluoride, stannoustartrate and the like. Stannous chloride (as SnCl₂.2H₂O) is mostpreferred. The reducing agent is present at a concentration of about 20to about 60 μg/mL, most preferably about 40 μg/mL.

The formulation of the present invention preferably contains one or morestabilizers chosen from maltose, ascorbic acid, gentisic acid orpharmaceutically acceptable salts of these acids. The sodium salt ofgentisic acid is most preferred. This reagent is preferably present at aconcentration of about 10 to about 25 mg/mL, most preferably 20 mg/mL.

An exchange ligand is preferably present in the formulation of thepresent invention to help stabilize Tc in a reduced oxidation stateuntil such time as it has chelated to the desired targeted chelatingligand. Such exchange ligands can include, for example, gentistic acid,gluconate, or glucoheptonate, or pharmacologically acceptable salts ofthese compounds. Sodium gluconate is most preferred. The exchange ligandis present at a concentration of about 1 to about 3 mg/mL, mostpreferably 1.3 mg/mL.

A detergent is optionally present in the formulation of the presentinvention to help improve recovery of the radioactive product from thevial. Such detergents can include non-ionic, cationic, anionic andzwitterionic surfactants including, for example, sodium dodecyl sulfate,N-dodecyl sulaine, Tween™ 80, cetyltrimethylammonium bromide,cyclo-n-methyl-β-D-malloside and n-hexyl-β-D-glucopyranoside. Non-ionicdetergents are particularly preferred, with cyclo-n-methyl-β-D-mallosideand n-hexyl-β-D-glucopyranoside especially preferred. Detergent may bepresent in an amount in the range of about 5-100 mg/ml, preferably about100 mg/ml.

Typical metal radionuclides used for diagnostic imaging agents include^(99m)Tc, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰⁹Pd, ¹⁹⁸Au, ¹⁹⁹Au, ¹¹¹In, in their variouschlorides, oxides or nitrides. Typical metal radionuclides used forradiotherapeutic applications include ¹⁸⁶Re, ¹⁸⁸Re, ¹¹¹In, ¹⁶⁶Ho, ¹⁰⁵Rh,¹⁴⁹Pm, ¹⁵³Sm, ¹⁷⁷Lu, ⁹⁰Y, ²⁰³Pb, ²¹²Pb, ²¹²Bi and the radioactiveactinides and additional radioactive lanthanides in their variouschlorides, oxides or nitrides. The preferred radioisotope used for thepreparation of radiopharmaceuticals made with the present formulationare isotopes of technetium and rhenium (e.g., ⁹⁴Tc, ^(99m)Tc, ¹⁸⁶Re,¹⁸⁸Re). Most preferably, the isotope used is ^(99m)Tc as a pertechnetatesalt, ^(99m)TcO₄ ⁻. Between about 5 and about 100 mCi of ^(99m)Tc can beadded, most preferably between about 20 and about 40 mCi.

The radiopharmaceutical formulation of the present invention can be usedto prepare radiopharmaceuticals that are reconstituted with radioisotopeat any temperature between room temperature and 100° C. When prepared atroom temperature, the optimal reaction time is between about 10 andabout 60 min, more preferably between about 15 and 30 minutes and mostpreferably for about 15 min. If prepared by heating to 100° C., optimalyield of the desired complex (>90% RCP) is obtained when kits are heatedfor about 5 minutes to about 30 min, most preferably between about 5 toabout 15 min.

Solubilization aids, such as cyclodextrins (e.g. alpha, beta or gammacyclodextrin or hydroxypropyl-γ-cyclodextrin) can optionally be presentin the formulations of the present invention. Gamma cyclodextrins arepreferred, and hydroxypropyl-γ-cyclodextrin is most preferred. Theconcentration of cyclodextrin may be from about 0.1 to about 10 mg/mL,preferably between about 2.5 and about 5 mg/mL.

As shown in FIG. 1, the radiopharmaceuticals of the invention have twoor three domains: a radiometal chelation moiety or chelating core, alinker or linking group (when present), and a receptor binding site ortargeting molecule. Together, the chelation moiety and targetingmolecule, with or without a linker, may be referred to as a chelatingligand. Compound 1, a preferred ligand for making radiopharmaceuticalformulations of the invention, has the following structure:

The synthesis of trifluoroacetic acid and acetic acid salts of Compound1 is described in Example 1. Example 2 describes the preparation ofCompound 1 complexed with ^(99m)Tc. In Example 3, the effect of thechelating core, namely the NMe₂-Gly-Ser-Cys portion of the ligand wasexamined by modifying the structure of this chelating core. Inparticular, Compounds 2-4 were synthesized as part of an experiment toexamine the effect of the chelating core on RCP:

The results, described in Example 3 and shown in Table 3, demonstratethat the radiopharmaceutical formulation containing Compound 1 exhibitssuperior RCP (i.e., greater than 90%), as compared to formulationscontaining Compounds 2-4.

Example 4 describes one embodiment of a kit for preparing aradiopharmaceutical formulation of the invention and its use forpreparing a technetium complex of Compound 1. Examples 5-12 describesynthesis and comparative testing of radiopharmaceutical compounds usingdetergents, rather than the diluent ethanol.

Targeting Molecules

Any molecule that specifically binds or reactively associates orcomplexes with a receptor or other receptive moiety associated with agiven target cell population may be used as a targeting molecule inradiopharmaceutical formulations of the invention. This cell reactivemolecule, to which the metal radionuclide chelation moiety is linkedoptionally via a linking group, may be any molecule that binds to,complexes with or reacts with the cell population sought to be bound orlocalized to. The cell reactive molecule acts to deliver theradiopharmaceutical to the particular target cell population with whichthe molecule reacts. The targeting molecule may be non-peptidic such as,for example, steroids, carbohydrates, lectins or small non-peptidicmolecules. The targeting molecule may also be an antibody, such as, forexample, a monoclonal or polyclonal antibody or a fragment thereof.Preferably the targeting molecule is a peptide, peptide mimetic orpeptoid. Most preferably the targeting molecule is a peptide. Peptideswhich are useful as targeting molecules include: bombesin, gastrin,gastrin-releasing peptide, transferrin, epidermal growth factors(“EGF”), platelet-derived growth factor, tumor growth factors (“TGF”),such as TGF-α and TGF-β, vaccinia growth factor (“VGF”), insulin andinsulin-like growth factors I and II, urotensin II peptides and analogs,NP-Y peptides, octreotide, depreotide, vapreotide, vasoactive intestinalpeptide (VIP), cholecystokinin (CCK), insulinlike growth factor (IGF),peptides targeting receptors which are upregulated in angiogenesis suchas VEGF receptors (e.g. KDR, NP-1, etc.), RGD-containing peptides,melanocyte-stimulating hormone (MSH) peptide, neurotensin, calcitonin,peptides from complementarity determining regions of an antitumorantibody, glutathione, YIGSR (leukocyte-avid peptides, e.g., P483H,which contains the heparin-binding region of platelet factor-4 (PF-4)and a lysine-rich sequence), atrial natriuretic peptide (ANP), β-amyloidpeptides, delta-opioid antagonists (such as ITIPP(psi)), annexin-V,endothelin, IL-1/IL-1ra, IL-2, IL-6, IL-8, leukotriene B4 (LTB4),chemotactic peptides (such asN-formyl-methionyl-leucyl-phenylalanine-lysine (fMLFK)), GP IIb/IIIareceptor antagonists (such as DMP444), epidermal growth factor, humanneutrophil elastase inhibitor (EPI-HNE-2, HNE2, and HNE4), plasmininhibitor, antimicrobial peptides, apticide (P280), P274, thrombospondinreceptor (including analogs such as TP-1300), bitistatin, pituitaryadenyl cyclase type I receptor (PAC1), and analogs and conservativesubstitutions of these peptides.

In preferred embodiments, the targeting molecule used in aradiopharmaceutical formulation of the invention is a biologicallyactive peptide. Most preferred embodiments of the present inventioninclude a peptide that binds to a gastrin releasing peptide receptor(GRP-r) and particularly a bombesin analog that is an agonist, such asthose disclosed in U.S. Pat. No. 6,200,546. Examples of such peptidesare shown in Table 1. Other bombesin analogs, fragments orpeptidomimetics may also be used as targeting molecules. For example,NAc-BBN[7-14] has been identified as the minimum fragment that retainsboth nanomolar binding affinity and elicits biological activity.However, there are a few specific amino acid substitutions in thebombesin 7-14 amino acid binding region (e.g. D-Ala¹¹ for L-Gly¹¹ orD-Trp⁸ for L-Trp⁸) which can be made without decreasing binding affinityor changing the compound from an agonist to an antagonist. For example,the presence of methionine (Met) at position BBN-14 will generallyconfer agonistic properties while the deletion of this residue generallyconfers antagonist properties [R. Camble, et al. Life Science (1989)45(17), 1521-7].

Furthermore, there is a tolerance to diverse functionalities whenchelates, solubilizing groups or linkers are attached at the N-terminusof the 7-14 amino acid residue of bombesin. When bombesin or a bombesinanalog is used as the biologically active peptide in aradiopharmaceutical formulation of the invention, theradiopharmaceutical may be targeted to tissue, particularly cancercells, that express GRP receptors.

TABLE 1 Amino Acid sequences of some natuxal bombesin-like peptides NameSequence Bombesin (BBN) PGlu-Gln-Arg-Leu-Gly- (SEQ ID NO:1)Asn-Gln-Trp-Ala-VaI- Gly-His-Leu-Met-NH₂ Alytesin PGlu-Gly-Arg-Leu-Gly-(SEQ ID NO:2) Thr-Gln-Trp-Ala-Val- Gly-His-Leu-Met-NH₂ H-GRPVal-Pro-Leu-Pro-Ala- (SEQ ID NO:3) Gly-Gly-Gly-Thr-Val-Leu-Thr-Lys-Met-Tyr- Pro-Arg-Gly-Asn-His- Trp-Ala-Val-Gly-His-Leu-Met-NH₂ Litorin PGlu-Gln-Trp-Ala-Val- (SEQ ID NO:4)Gly-His-Phe-Met-NH₂ Ranatensin PGlu-Val-Pro-Gln-Trp- (SEQ ID NO:5)Ala-Val-Gly-His-Phe- Met-NH₂ Human Ala-Pro-Leu-Ser-Trp- (SEQ ID NO:6)neuromedin B Asp-Leu-Pro-Glu-Pro- Arg-Ser-Arg-Ala-Ser-Lys-Ile-Arg-Val-His- Ser-Arg-Oly-Asn-Leu- Trp-Ala-Thr-Gly-His-Phe-Met-NH₂ Phyllolitorin PGlu-Leu-Trp-Ala-Val- (SEQ ID NO:7)Gly-Ser-Phe-Met-NH₂Linking Groups

The terms “spacer,” “spacer group,” “linker,” and “linking group” areused synonymously herein to refer to a chemical group that serves tocouple the targeting molecule to the metal chelator while not adverselyaffecting either the targeting function of the targeting molecule or themetal complexing function of the metal chelator. Suitable spacer groupsinclude peptides (i.e., amino acids linked together) alone, anon-peptide group (e.g., hydrocarbon chain) or a combination of an aminoacid sequence and a non-peptide spacer. A pure peptide spacer consistsof a series of amino acids (e.g., diglycine, triglycine, Gly-Gly-Glu,Gly-Ser-Gly, etc.), in which the total number of atoms between theN-terminal residue of the BBN binding moiety and the metal chelator inthe polymeric chain is less than or equal to 12 atoms.

The spacer may also include a hydrocarbon chain (i.e.,—R₁—(CH₂)_(n)—R₂—) wherein n is 0-10, preferably n=3 to 9, R₁ is aderivative of a group (e.g., H₂N—, HS—, —COOH) that can be used as asite for covalently linking the ligand backbone or the preformed metalchelator or metal complexing backbone; and R₂ is a group that is usedfor covalent coupling to the N-terminal NH₂-group of the methods forconjugating ligands (i.e., chelators) or preferred metal chelates tobiomolecules have been described in the literature. See e.g., Wilbur(1992) Bioconj. Chem., 3: 433; Parker (1990) Chem. Soc. Rev., 19: 271;Hermanson (1996) In: Bioconjugate Techniques, Academic Press, pp. 3-136;Fritzberg et al. (1995) Radiolabeling of antibodies for targeteddiagnostics. In: Targeted Delivery of Imaging Agents (ed) V. P.Torchilin, CRC Press, Boca Raton, Fla., pp. 84-101 (each of which ishereby incorporated by reference in its entirety). One or more of thesemethods may be used to link either the uncomplexed ligand (chelator) orthe radiometal chelate to the spacer group or to link the spacer groupto the targeting molecule. These methods include the formation of acidanhydrides, aldehydes, arylisothiocyanates, activated esters, orN-hydroxysuccinimides. A preferred linking group is -Gly-5-aminovalericacid-. Additional linkers suitable for use in the subject invention aredisclosed in copending patent application U.S. Ser. No. 60/439,722,filed Jan. 13, 2003 and U.S. Ser. No. 10/341,577, filed Jan. 13, 2003,the entire contents of which are hereby incorporated by reference.

Preparation of Radiopharmaceuticals and the Other Formulation Components

The present invention also provides stabilized formulations ofradiopharmaceuticals containing the appropriate and usual additives suchas buffers, bulling agents, etc. In another embodiment, the presentinvention may also include single and/or multi-dose kits for preparingradiopharmaceuticals using the formulations of the invention. Allnon-radioactive reagents of the formulation may be formulated togetherin a single vial or the complexing ligand may be present in one vialwhile the stannous or other reducing source may be present in a secondvial. In another embodiment, the kit may include a transfer (ortrans-chelating) ligand and stannous or other reducing agent in thefirst vial and the complexing ligand in the second vial. In a furtherembodiment, the kit formulations may include the usual additives andbulking agents known to those skilled in the art. Methods ofreconstituting the kits with radioisotopes are well known to thoseskilled in the art.

Kits of the present invention comprise one or more vials containing thesterile formulation of a predetermined amount of a complexing ligand,and optionally other components such as reducing agents, transferligands, buffers, lyophilization aids or bulking agents, stabilizationaids, solubilization aids, bacteriostats and diluents. The inclusion ofone or more optional components in the formulation will frequentlyimprove the ease of synthesis of the radiopharmaceutical by thepracticing end user, the ease of manufacturing the kit, the shelf-lifeof the kit, or the stability and shelf-life of the radiopharmaceutical.The improvement achieved by the inclusion of an optional component inthe formulation must be weighed against the added complexity of theformulation and added cost to manufacture the kit. The one or more vialsthat contain all or part of the formulation can independently be in theform of a sterile solution or a lyophilized solid.

Buffers useful in the preparation of radiopharmaceuticals and indiagnostic kits useful for the preparation of the radiopharmaceuticalsinclude but are not limited to phosphate, citrate, sulfosalicylate, andacetate. A more complete list can be found in the United StatesPharmacopeia.

Lyophilization aids or bulking agents may optionally be used in theradiopharmaceutical formulations of the invention. Lyophilization andbulking agent are known in the art and include mannitol, lactose, sodiumchloride, maltose, sucrose, PEG 8000, cyclodextrins, such ashydroxypropyl-γ-cyclodextrin (HP-γ-CD), dextran, Ficoll, andpolyvinylpyrrolidine (PVP). Of these, sodium chloride, maltose, HP-γ-CD,and dextran are preferred bulking agents for use with the invention,with HP-γ-CD being the most preferred.

Stabilization aids or stabilizers useful in the radiopharmaceuticalformulations of the invention include but are not limited to ascorbicacid, para-aminobenzoic acid (PABA), sodium bisulfite, sodiummetabisulfite, gentisic acid, t-butanol and inositol. Gentisic acid isthe most preferred stabilization aid.

Solubilization aids useful in the preparation of radiopharmaceuticalsand in diagnostic kits useful for the preparation of theradiopharmaceuticals include but are not limited to ethanol, glycerin,polyethylene glycol, propylene glycol, polyoxyethylene sorbitanmonooleate, sorbitan monooloeate, polysorbates,poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers(Pluronics) and lecithin. The preferred solubilizing aid is ethanol.

As noted above, a detergent is optionally present in the formulation ofthe present invention to help improve recovery of the radioactiveproduct from the vial. Such detergents can include non-ionic, cationic,anionic and zwitterionic surfactants including, for example, sodiumdodecyl sulfate, N-dodecyl sulaine, Tween™ 80, cetyltrimethylammoniumbromide, cyclo-n-methyl-β-D-malloside and n-hexyl-β-D-glucopyranoside.The latter two components are especially preferred. Detergent may bepresent in an amount in the range of about 5-100 mg/ml, preferably about100 mg/ml.

Bacteriostats useful in the preparation of radiopharmaceuticals and indiagnostic kits useful for the preparation of the radiopharmaceuticalsinclude but are not limited to benzyl alcohol, benzalkonium chloride,chlorbutanol, and methyl, propyl or butyl parabens.

A component in a diagnostic kit can also serve more than one function. Areducing agent can also serve as a stabilization aid, a buffer or astabilizer can also serve as a transfer ligand, a lyophilization aid canalso serve as a transfer, ancillary or co-ligand and so forth.

In a preferred embodiment the kit of the invention includes: a targetedchelating ligand of the structure shown in Formula I; a stannous saltsuch as stannous chloride, fluoride or tartrate; a stabilizing aid suchas gentisic acid or a pharmaceutically acceptable salt thereof; anexchange ligand such as gluconic acid or a pharmaceutically acceptablesalt thereof; and a solubilizing agent such as ethanol and/or acyclodextrin such as hydroxypropyl gamma cyclodextrin. Most preferably,the kit includes one or two vials. In a particularly preferredembodiment, the kit includes two vials, the first vial containing 4 μgof Compound 1 (triflate or acetate salt); 40 μg of SnCl₂.2H₂O; 1.3 mg ofGluconic acid, sodium salt; 20 mg of gentisic acid, sodium salt; andoptionally 2.5 mg of hydroxypropyl-γ-cyclodextrin, with the pH of theformulation adjusted to a pH of about pH 3-4 using a pharmacologicallyacceptable acid or base such as HCl or NaOH; the second vial containsethanol.

The following abbreviations are used in the examples: TFA,trifluoroacetic acid; HOBt, 1-hydroxybenzotriazole; DIC,N,N′-diisopropylcarbodiimide; HATU,O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; DIEA, diisopropylethylamine; DMF,dimethylformamide; DMSO, methyl sulfoxide; CH₂Cl₂, methylene chloride;EtOH, ethanol. RCP, radiochemical purity; HPLC, high pressure liquidchromatography. Unless otherwise noted, all materials were purchasedfrom Aldrich and used without further purification. Fmoc-5-amino-valericacid (5-ava) was purchased from Chem-Impex International. All Fmoc-aminoacids were purchased from Novabiochem.

EXAMPLE 1 Solid Phase Peptide Synthesis of Compound 1

Compound 1 was prepared as both its TFA and acetate salt using solidphase peptide synthesis (SPPS), following the procedure described below.Unless otherwise noted, all materials were purchased from Aldrich andused without further purification.

Synthesis of the TFA Salt of Compound 1:

As shown in Scheme 1 above, synthesis of the compound was carried out indimethyl formamide (DMF) using HOBt/DIC activation on rink-amide Novagelresin. Fmoc deprotection was carried out with 20% piperidine in DMF. Theresin was swelled in DMF for 1 h before use. All couplings were of 2hours duration except for the last N,N-dimethylglycine coupling (seebelow). A ninhydrin test was employed to determine reaction completion.

A typical coupling cycle is as follows: To a 50-mL SPPS reaction vesselcontaining 1.13 mmol of the swelled resin (0.6 mmol/g, Novabiochem) wasadded a solution of 4.52 mmol of an Fmoc-amino acid in DMF (EM Science),4.52 mmol of HOBT (Novabiochem) in DMF, and 4.52 mmol of DIC. The totalvolume of DMF was 20 mL. The reaction mixture was shaken for 2 h. Theresin then was filtered and washed with DMF (3×30 mL). A ninhydrin testwas carried out to confirm the completion of the coupling. A solution of20% piperidine in DMF (20 mL) was added to the resin and it was shakenfor 10 min. The resin was filtered and this piperidine treatment wasrepeated. The resin was finally washed with DMF (3×30 mL) in preparationfor the next coupling cycle.

At the last coupling cycle, N,N-dimethyl glycine was coupled usingHATU/DIEA activation. Thus, to a suspension of N,N-dimethyl glycine(4.52 mmol) in DMF was added a solution of 4.52 mmol of HATU (PerseptiveBiosystems) in DMF and 9.04 mmol of DIEA. The clear solution was addedto the resin and shaken for 16 h. Following synthesis, the resin waswashed with DMF (3×30 mL) and CH₂Cl₂ (3×30 mL). It was dried by blowingN₂ through the container for 15 min. 30 mL of reagent B

(TFA/phenol/H₂O/triisopropylsilane/methyl sulfide 86 mL/5 g/5 mL/2 mL/2mL) was added and it was shaken for 4 h. The resin was filtered and thefiltrate was evaporated to a paste. The crude peptide was precipitatedin diethyl ether and washed twice with ether. 1.2 g of the crudematerial was obtained after drying. The peptide was purified usingShimadzu HPLC system and a YMC C-18 preparative column. Crude materialwas dissolved in 15% CH₃CN/H₂O (0.1% TFA) and loaded on the column. Thegradient consisted of an increase from 15% to 19% CH₃CN/H₂O (0.1% TFA)in 4 min., followed by 19% to 49% in 60 min. The fractions were combinedand lyophilized. A total of 840 mg of the pure material was obtained.

Synthesis of the Acetate Salt of Compound 1:

The TFA salt of Compound 1 (110 mg in 20 mL of 0.5 M HOAc) was loadedonto a 30-mL AG 1×2 ion exchange resin column (acetate form, Bio-RadLaboratories). The resin was pre-washed with water until a constantconductivity was obtained. It was eluted with 0.5 M HOAc at a 12 mL/minflow rate. The desired fractions were combined and lyophilized. Cautionwas taken to avoid prolonged exposure of the material to air. 90 mg ofthe acetate salt of Compound 1 was obtained.

EXAMPLE 2 Formulation for the Preparation of Compound 1 Complexed with^(99m)Tc

A solution of the TFA salt of Compound 1 synthesized as described inExample 1 was prepared at a concentration of 0.08 mg/mL in 0.05N HCl or0.1% TFA. A pH 3 solution of gentisic acid was prepared by dissolvinggentisic acid, sodium salt (Sigma) to a concentration of 100 mg/mL andthe pH was adjusted to between 2.9 and 3.0 with HCl (1N). A stannouschloride solution (20 mg/mL) was prepared by dissolving SnCl₂.2H₂O inN₂-purged HCl (1N). A stannous gluconate solution was prepared by theaddition of 20 μL of stannous chloride solution to 1 mL of sodiumgluconate solution. A hydroxypropyl-γ-cyclodextrin solution (50 mg/mL)was prepared by dissolving HP-γ-CD in water. All the solvents used toprepare these solutions were purged with nitrogen.

To prepare the ^(99m)Tc complex of Compound 1, 300 μL of ethanol, 50 μLof ligand solution (4 μg Compound 1), 200 μL of gentisic acid solution(20 mg), 50 μL of hydroxypropyl-γ-cyclodextrin solution (2.5 mg) 100 μLof stannous gluconate solution (40 μg of SnCl₂), and ^(99m)TcO₄ ⁻ (˜40mCi) was added. Water was added to bring the final volume to 1.0 mL, andthe reaction was heated at 100° C. for 15-20 min.

The ^(99m)Tc labeled peptide purity was determined by HPLC using a VydacC18 protein and peptide analytical column, 300 A pore size, 4.6×250 mmcolumn length. The following gradient (flow rate 1 mL/min) was used:Isocratic elution 78% H₂O (0.1% TFA)/22% CH₃CN (0.1% TFA) for 20 min,gradient from 78% to 40% CH₃CN (0.1% TFA) over 5 min, hold at 40% CH₃CN(0.1% TFA) for 10 min, ramp from 40% to 78% CH₃CN (0.1% TFA) over 5 min.

The percentage of radiocolloid in the radiolabeled product wasdetermined using a C18 (10×2 cm) plate eluted with

60% CH₃CN/30% H₂O/10% NH₄OH. A 2 μL aliquot of the reaction mixture werespotted at 1.5 cm and the plate developed up to 9.5 cm. The plates werecounted on a Bioscan Radiochromatoscan, Model AR2000. The activitymeasured from 0 to 2 cm represents the % radiocolloid. The formulationprepared in this example had a radiochemical purity (RCP) value of ˜93%at 18 minutes and 91% at 6 hours post reconstitution with 40 mCi of^(99m)TcO₄ ⁻.

EXAMPLE 3 Effect of Chelating Core on RCP

The following experiment was performed to examine the effect of thechelating core on RCP. The TFA salt of Compound 1((NMe₂-Gly-Ser-Cys)-Gly-(5-aminovalericacid)-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂) was synthesized as describedin Example 1. The compounds shown in Table 2 were prepared by solidphase peptide synthesis following appropriate modification of thesynthesis shown in Example 1.

TABLE 2 Structure of Additional Compounds used in Example 3 Cmpd. No.Structure 2 NMe₂-Gly-Gly-Cys-Gly-5-aminovaleroyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-MH₂ 3NH₂-Gly-Ser-Cys-Gly-5-aminovalerioyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ 4NH₂-Gly-Gly-Cys-Gly-5-aminovaleroyl-Gln-Trp-Ala-Val-Gly- His-Leu-Met-NH₂

Solutions of Compounds 1-4 were prepared at a concentration of 0.08mg/mL in 0.05 N HCl. A pH 3 solution of gentisic acid was prepared bydissolving gentisic acid, sodium salt (223 mg, Sigma) in water and thepH was adjusted to 3.0 with HCl (1N). The final volume of the solutionwas 2.79 mL. A stannous chloride solution was prepared by dissolvingSnCl₂.2H₂O (1.547 mg, 0.007 mmol, Aldrich) in 500 μL of N₂ purged 0.05NHCl. To this solution was added 51 mg (0.23 mmol) of gluconic acid,sodium salt (Sigma) and the volume was adjusted to 2978 μL. All solventsused to prepare these solutions were purged with nitrogen.

To prepare the ^(99m)Tc complex, 50 μL of ligand solution (4 μg Compound1-4), 300 μL of EtOH, 250 μL of gentisic acid solution (20 mg), 50 μL of1SnCl₂: 34 Gluconic acid (40 μg of SnCl₂), and ^(99m)TcO₄ ⁻ (˜40 mCi,Syncor) was added. Water was added to bring the final volume to 1.0 mL,and the reaction was allowed to stand at room temperature for 15 min.The solutions were then analyzed for RCP as described in Example 2. Theresults are shown in Table 3.

TABLE 3 HPLC Analysis of Compounds 1-4, Average RCP Values Percent RCPPercent RCP Percent Compound at 15 min at 6 hours Radiocolloid 1 91.288.1 1.2 2 80.8 78.7 0.95 3 86.7 82.4 0.31 4 83.3 83.3 2Each experiment was performed multiple times and the average is reportedin Table 3.

EXAMPLE 4 Kits for the Preparation ofTcOMe₂N-Gly-Ser-Cys-Gly-5-aminovaleroyl-Glu-Trp-Ala-Val-Gly-ylis-Leu-Met-NH₂

This example describes a two component kit used for the preparation of aradiopharmaceutical of the invention. As summarized in Table 4, the kitmay include two vials, however the contents of Vial 2 need not besupplied with the kit.

TABLE 4 A kit for the preparation of a radiopharmaceutical formulationof the invention Vial 1 Vial 2 4 μg Compound 1 (triflate or acetatesalt) 300 μL of EtOH 40 μg of SnCl₂2H₂O 1.3 mg of Gluconic acid, sodiumsalt 20 mg of gentisic acid, sodium salt 2.5 mg ofhydroxypropyl-γ-cyclodextrin (optional) pH 3-4

Vial 1 is reconstituted with 300 μL of EtOH from Vial 2, 40 mCi of^(99m)TcO₄ ⁻ is added, followed by normal saline to a final volume of1.0 mL. The vial is then either allowed to stand at room temperature for15 min., or heated at 100° C. for 5 to 20 min. As indicated in Table 4,the inclusion of hydroxypropyl-γ-cyclodextrin is optional.

Use of Detergent in Lyophilized Formulation

The previously described formulation developed for the syntheses of^(99m)Tc-COMPOUND 1 comprises 30% (300 μl) EtOH, which is utilized toimprove recovery of the ^(99m)Tc complex from the vial. If afreeze-dried kit is desired, EtOH, being a liquid, requires a two-vialformulation because it cannot be freeze-dried with the other formulationcomponents. The use of a solid compound instead of EtOH would reduce thenumber of vials from two to one, thus potentially reducing productioncosts considerably.

Two non-ionic detergents, Cyclohexyl-n-methyl-β-D-maltoside andn-Hexyl-β-D-Glucopyranoside, were tested for this purpose.

Cyclohexyl-n-methyl-β-D-maltoside n-Hexyl-β-D-Glucopyranoside

These detergents were chosen because they should not dissolve biologicalmembranes (they are not ionic detergents), they are very water-soluble,and they contain a sugar moiety that has potential exchange ligandproperties.

EXAMPLE 5 Syntheses of ^(99m)Tc COMPOUND 1 (No ethanol or surfactantadded)

A stannous gluconate solution was prepared by dissolving 2.303 mg (0.001mmol) of SnCl₂.2H₂O in 500 μL of N₂-purged 0.05N HCl. To this solutionwas added 76 mg (0.347 mmol) of sodium gluconate and the volume wasadjusted to 2.878 mL of water.

Compound 1 (275 μg, COMPOUND 1 acetate salt) was dissolved in 2.750 mLof 0.05N HCl. Gentisic acid (158.6 mg) was dissolved in water and the pHwas adjusted to 3.0 using 1N HCl; the final volume was 2.08 mL.

To 50 μL of the Compound 1 solution (4 μg) was added ^(99m)TcO₄ ⁻ (190μL, 41.5 mCi), 0.458 mL of H₂O, 50 μL of the stannous gluconate solution(40 μg of SnCl₂) and 262 μL of gentisic acid solution (20 mg). Thereaction was heated at 100° for 5 min. prior to HPLC analysis, which wasperformed using the following system: Vydac C-18 Protein and Peptidecolumn (4.6×250 mm) eluted with 78% H₂O (0.1% TFA)/22% CH₃CN (0.1% TFA)for 20′, ramped from 78% to 40% H₂O (0.1% TFA) in 5′, held at 40% H₂O(0.1% TFA) for 10′, then ramped from 40% to 78% H₂O (0.1% TFA) in 5′.Flow rate: 1 mL/min. The resulting RCP was 87.4% initially and 87.1% at6 hours. The recovery from the vial was 67%.

This example shows that recovery of ^(99m)Tc COMPOUND 1 from the vialwas low in the absence of added ethanol or surfactant.

EXAMPLE 6 Syntheses of ^(99m)Tc COMPOUND 1 with 300 μl of EtOH (nosurfactant)

Stannous gluconate, Compound 1 and Gentisic acid solutions were preparedas described in Example 5. To 50 μL of the Compound 1 solution (4 μg)was added ^(99m)TcO₄ ⁻ (170 μL, 38.5 mCi), 0.178 mL of H₂O, 50 μL of thestannous gluconate solution (40 μg of SnCl₂), 262 μL of gentisic acidsolution (20 mg) and 300 μL of EtOH. The reaction was heated andanalyzed as described in Example 5. The resulting RCP ranged from 91.1to 93.1%, and ranged from 87.0 to 90.3% at 6 hours. Radiocolloid valuesof 0.5 and 0.7 were observed (n=2) The recovery from the vial was 98.44%(n=1).

This example shows that recovery of ^(99m)Tc COMPOUND 1 from the vialcould be improved by substitution of EtOH for some of the water in theformulation.

EXAMPLE 7 Syntheses of ^(99m)Tc COMPOUND 1 with 10 mg ofCyclohexyl-n-methyl-β-D-maltoside:

A stannous gluconate solution was prepared by dissolving 1.597 mg (0.007mmol) of SnCl₂.2H₂O in 500 μL of N₂-purged 0.05N HCl. To this solutionwas added 51 mg (0.233 mmol) of sodium gluconate and the volume wasadjusted to 1.996 mL with water. Compound 1 (211 μg, COMPOUND 1 acetatesalt) was dissolved in 2.637 mL of 0.05N HCl. Gentisic acid (238.8 mg)was dissolved in water and the pH was adjusted to 3.0 using 1N HCl; thefinal volume was 3.085 mL. Cyclohexyl-n-methyl-β-D-maltoside (55 mg) wasdissolved in 550 μL of H₂O.

To 50 μL of the Compound 1 solution (4 μg) was added ^(99m)TcO₄ ⁻ (270μL, 38 mCi), 0.272 mL of H₂O, 50 μL of the stannous gluconate solution(40 μg of SnCl₂), 258 μL of gentisic acid solution (20 mg) and 100 μL(10 mg) of the maltoside solution. The reaction was heated and analyzedas described in Example 5. The resulting RCP was 88.2% and the % ofradiocolloid was 1.3%.

This example shows that the yield of ^(99m)TcO COMPOUND 1 was notaffected by the addition of maltoside, when compared to that obtained inits absence (Example 5).

EXAMPLE 8 Syntheses of ^(99m)Tc COMPOUND 1 with 15 mg ofCyclohexyl-n-methyl-β-D-maltoside

Stannous gluconate, Compound 1 and Gentisic acid solutions were preparedas described in Example 5. Cyclohexyl-n-methyl-β-D-maltoside (36.2 mg)was dissolved in 518 μL of H₂O. To 50 μL of the Compound 1 solution (4μg) was added ^(99m)TcO₄ ⁻ (210 μL, 38.4 mCi), 0.338 mL of H₂O, 50 μL ofthe stannous gluconate solution (40 μg of SnCl₂), 262 μL of gentisicacid solution (20 mg) and 100 μL (15 mg) of the maltoside solution. Thereaction was heated and analyzed as described in Example 5. Theresulting RCP was 88.0% and recovery from the vial was 90.3%.

This example shows that relative to the conditions used in Example 5,recovery of ^(99m)Tc COMPOUND 1 from the vial could be improved byaddition of Cyclohexyl-n-methyl-β-D-maltoside to the vial, withoutadversely affecting RCP.

EXAMPLE 9 Syntheses of ^(99m)Tc COMPOUND 1 with 50 mg ofCyclohexyl-n-methyl-β-D-maltoside

Stannous gluconate, Compound 1, and gentisic acid solutions wereprepared as described in Example 7. Cyclohexyl-n-methyl-β-D-maltoside(85 mg) was dissolved in 510 μL of H₂O. To 50 μL of the Compound 1solution (4 μg) was added ^(99m)TcO₄ ⁻ (230 μL, 39.6 mCi), 0.168 mL ofH₂O, 50 μL of the stannous gluconate solution (40 μg of SnCl₂), 262 μLof gentisic acid solution (20 mg) and 300 μL (50 mg) of the maltosidesolution. The reaction was heated and analyzed as described in Example5. The resulting RCP was 88.5 and 90.5%. The recovery from the vial was95.6% (n=1).

This example shows that relative to the conditions used in Example 5,recovery of ^(99m)Tc COMPOUND 1 from the vial could be improved byaddition of Cyclohexyl-n-methyl-β-D-maltoside to the vial, withoutadversely affecting RCP, and that recovery was improved by the use of 50mg of surfactant, relative to the use of 15 mg of surfactant.

EXAMPLE 10 Syntheses of ^(99m)Tc COMPOUND 1 with 100 mg ofCyclohexyl-n-methyl-β-D-maltoside

Stannous gluconate, Compound 1 and Gentisic acid solutions were preparedas described in Example 7. Cyclohexyl-n-methyl-β-D-maltoside (127.9 mg)was dissolved in 518 μL of H₂O. To 50 μL of the Compound 1 solution (4μg) was added ^(99m)TcO₄ ⁻ (220 μL, 41.6 mCi), 0.022 mL of H₂O, 50 μL ofthe stannous gluconate solution (40 μg of SnCl₂), 258 μL of gentisicacid solution (20 mg) and 400 μL (10 mg) of the maltoside solution. Thereaction was heated and analyzed as described in Example 5. Theresulting RCP was 90%. The % of radiocolloid was 0.5%.

EXAMPLE 11 Syntheses of ^(99m)Tc COMPOUND 1 with 10 mg ofn-Hexyl-β-D-Glucopyranoside:

A stannous gluconate solution was prepared by dissolving 2.383 mg (0.001mmol) of SnCl₂ ⁻2H₂O in 500 μL of N₂-purged 0.05N HCl. To this solutionwas added 78 mg (0.36 mmol) of sodium gluconate and the volume wasadjusted to 2.978 mL with water. Compound 1 (265 μg) (acetate salt) wasdissolved in 2.650 mL of 0.05N HCl. Gentisic acid (204.1 mg) wasdissolved in water and the pH was adjusted to 3.0 using 1N HCl; thefinal volume was 2.551 mL. n-Hexyl-β-D-Glucopyranoside (180.6 mg) wasdissolved in 672 μL of H₂O.

To 50 μL of the Compound 1 solution (4 μg) was added ^(99m)TcO₄ ⁻ (210μL, 38 mCi), 0.41 mL of H₂O, 50 μL of the stannous gluconate solution(40 μg of SnCl₂), 250 μL of gentisic acid solution (20 mg) and 40 μL (10mg) of the maltoside solution. The reaction was heated and analyzed asdescribed in Example 5. The resulting RCP was 86.4% and the % ofradiocolloid was 1.1%. Recovery from the vial 96.3%.

This example shows that relative to the conditions used in Example 5,recovery of ^(99m)Tc COMPOUND 1 from the vial could be improved byaddition of n-Hexyl-β-D-Glucopyranoside to the vial, without adverselyaffecting RCP.

EXAMPLE 12 Syntheses of ^(99m)Tc COMPOUND 2 with 50 mg ofn-Hexyl-β-D-Glucopyranoside:

Stannous gluconate, Compound 1 and Gentisic acid solutions were preparedas described in Example 11. n-Hexyl-β-D-Glucopyranoside (180.6 mg) wasdissolved in 672 μL of H₂O.

To 50 μL of the Compound 1 solution (4 μg) was added ^(99m)TcO₄ ⁻ (160μL, 41 mCi), 0.3 mL of H₂O, 50 mL of the stannous gluconate solution (40μg of SnCl₂), 250 μL of gentisic acid solution (20 mg) and 200 μL (50mg) of the maltoside solution. The reaction was heated and analyzed asdescribed in Example 5. The resulting RCP was 92.7% initially, and 89.1%at 6 hours. The % of radiocolloid was 1.2% and the recovery from thevial 98.8%. A representative chromatogram is shown below.

SUMMARY

As it is shown in the following table the recoveries obtained usingthese detergents are very similar to the ones in which EtOH was used.Moreover the RCP obtained with n-Hexyl-β-D-Glucopyranoside arecomparable to the ones with EtOH.

Initial RCP 6 h Colloids Detergent RCP (%) (%) (%) Recovery (%) No EtOH87.4 87.1 67.0 EtOH 93.1 88.2 98.4 EtOH 92.5 90.3 0.7 EtOH 91.1 87.0 0.598.6 10 mg maltoside 88.2 1.3 15 mg maltoside 88.0 90.3 50 mg maltoside88.5 95.6 50 mg maltoside 90.5 85.2 1 100 mg maltoside 90.0 0.5 10 mgGlucopyrano- 86.4 1.1 96.3 side 50 mg Glucopyrano- 90.8 0.4 side 50 mgGlucopyrano- 92.7 89.1 1.2 98.8 side

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

1. A radiopharmaceutical composition comprising: a compound of Formula1a

a reducing agent; an exchange ligand; a stabilizer; a detergent; adiluent; and a radionuclide, wherein L is optional and is a linkinggroup; Z is a targeting molecule; and wherein the compound of Formula 1ais present at less than 10 μg per mL of diluent.
 2. A composition forthe preparation of a radiopharmaceutical comprising: a compound ofFormula 1a

a reducing agent; an exchange ligand; a stabilizer; and a detergentwherein L is optional and is a linking group; Z is a targeting molecule,and wherein the compound of Formula 1a is present in an amount such thatthe resulting radiopharmaceutical contains less than 10 μg per mL ofdiluent.
 3. A composition of any one of claims 1 or 2 wherein Z is abombesin analog.
 4. A composition of claim 3 wherein Z is selected fromthe group consisting of SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ IDNO:4; SEQ ID NO:5; SEQ ID NO:6; and SEQ ID NO:7.
 5. Aradiopharmaceutical composition comprising: a compound of Formula 2

a reducing agent; an exchange ligand; a stabilizer; a detergent; adiluent; and a radionuclide, wherein the compound of Formula 2 ispresent at less than 10 μg per mL of diluent.
 6. A composition for thepreparation of a radiopharmaceutical comprising: a compound of Formula 2

a reducing agent; an exchange ligand; and a stabilizer; and a detergent,wherein the compound of Formula 2 is present in an amount such that theresulting radiopharmaceutical contains less than 10 μg per mL of adiluent.
 7. A composition of any one of claims 2 or 6 wherein thereducing agent is selected from the group consisting of stannous salt,sodium borohydride, cupric salts, and formamidine sulfinic acid.
 8. Acomposition of any one of claims 2 or 6 wherein the exchange ligand isselected from the group consisting of gluconate, gentisic acid andglucoheptonate.
 9. A composition of any one of claims 2 or 6 wherein thestabilizer is selected from the group consisting of gentisic acid,maltose and ascorbic acid.
 10. A composition of any one of claims 2 or 6further comprising a solubilizer.
 11. A composition of claim 10, whereinthe solubilizer is a cyclodextrin.
 12. A composition of claim 11,wherein the solubilizer is hydroxypropyl-γ-cyclodextrin.
 13. Acomposition of any one of claims 1 or 5 wherein the diluent is selectedfrom the group consisting of ethanol, water and saline.
 14. Aradiopharmaceutical composition comprising: a compound of Formula 2

stannous chloride; gluconic acid; gentisic acid; ethanol; and aradionuclide, wherein the compound of formula 2 is present at less than10 μg per mL of diluent.
 15. A composition for the preparation of aradiopharmaceutical comprising: a compound of Formula 2,

stannous chloride; gluconic acid; and gentisic acid, wherein thecompound of Formula 2 is present in an amount such that the resultingradiopharmaceutical contains less than 10 μg per mL of a diluent.
 16. Acomposition of claim 15, further comprisinghydroxypropyl-γ-cyclodextrin.
 17. A radiopharmaceutical composition ofclaim 1, wherein the compound of Formula 1 is present at less than 5 μgper mL of diluent.
 18. A radiopharmaceutical composition of any one ofclaims 1 or 5, wherein the radiopharmaceutical is prepared andadministered to a subject without purification.
 19. Aradiopharmaceutical composition of any one of claims 1 or 5, wherein theRCP of the composition is greater than or equal to about 90% at about 15minutes.
 20. A composition of any one of claims 2 or 6, wherein thecompound of Formula 1 is present in an amount such that the resultingradiopharmaceutical contains less than 5 μg per mL of diluent.
 21. Acomposition of any one of claims 2 or 6, wherein the resultingradiopharmaceutical is prepared and administered to a subject withoutpurification.
 22. A composition of any one of claims 2 or 6, wherein theRCP of the resulting radiopharmaceutical is greater than or equal toabout 90% at about 15 minutes.
 23. A composition of claim 15, furthercomprising a detergent.
 24. A composition of claim 15, furthercomprising a detergent, wherein the composition is in lyophilized form.25. A composition of any one of claims 2, 6, or 15 further comprising adiluent, optionally contained in a separate container.
 26. Theradiopharmaceutical of claim 14 further comprising a detergent.
 27. Theradiopharmaceutical of any one of claims 1, 5 or 26 wherein thedetergent is selected from the group consisting of non-ionic, cationicand zwitterionic surfactants.
 28. The composition of any one of claims2, 6 or 23 wherein the detergent is selected from the group consistingof non-ionic, cationic and zwitterionic surfactants.
 29. Theradiopharmaceutical of any one of claims 1, 5 or 26 wherein thedetergent is a non-ionic detergent.
 30. The composition of any one ofclaims 2, 6 or 23, wherein the detergent is selected from the groupconsisting of cyclo-n-methyl-β-D-maltoside andn-hexyl-β-D-glucopyranoside.
 31. The radiopharmaceutical of any one ofclaims 1, 5 or 26, wherein the detergent is selected from the groupconsisting of cyclo-n-methyl-β-D-maltoside andn-hexyl-β-D-glucopyranoside.